S. Savocchia, R. Billones-Baaijens, A. Cimmino, A. Evidente Isolation and characterisation of phytotoxins produced by Botryosphaeriaceae and their role in grapevine trunk diseases PIERLUIGI REVEGLIA PhD Candidate Supervisors: S. Savocchia, R. Billones-Baaijens, A. Cimmino, A. Evidente

Botryosphaeria dieback (BD) INTRODUCTION Botryosphaeria dieback (BD) A serious problem of grapevines worldwide Ranked in the top five priority diseases of the Australian wine grape industry Caused by species of fungi – Botryosphaeriaceae 11 species reported in Australian vineyards Causes cankers, dieback and eventually death of vines Photo: W. Pitt

Photo: L. Mugnai In Europe, the disease also produces foliar symptoms associated with phytotoxins produced by BD pathogens

No foliar symptoms have been observed in Australian vineyards to date Investigate the role of phytotoxins in the pathogenicity and symptom expression of Botryosphaeria dieback pathogens in Australian vineyards

RESEARCH QUESTIONS Do Australian Botryosphaeriaceae spp. produce phytotoxins in vitro and in planta? Are these phytotoxins the same/different from those reported in other countries? Are the phytotoxins involved in pathogenicity and virulence of these pathogens? What is their role in the symptomatology observed in vineyards? What gene families are involved in phytotoxin production?

OBJECTIVE 1: Identify and biologically and chemically characterise the phytotoxic metabolites produced by Botryosphaeriaceae spp. Botryosphaeria spp. Botryosphaeria dothidea Diplodia mutila Diplodia seriata Dothiorella vidmadera Dothiorella sp. Neofusicoccum australe Neofusicoccum luteum Neofusicoccum parvum Spencermartinsia viticola Liquid cultures Liquid extraction TLC analysis Grapevine leaf assays

Production of phytotoxic metabolites PURIFICATION CHARACTERISATION Silica gel column Preparative TLC 1D and 2D NMR spectroscopy ESI/LC MS Reveglia et al., Natural Product Research, 2018 (accepted)

Phytotoxins from Spencermartinsia viticola Reveglia et al., Arab. J. Chem., 2018 doi.org/10.1016/j.arabjc.2018.01.014.

Phytotoxins from Dothiorella vidmadera Reveglia et al., J. Agr. Food Chem., 2018, 66, 1760-1764.

FIRST CONCLUSIONS: ….WHAT’S NEXT? The most virulent and widespread Australian Botryosphaeriaceae spp. are capable to produce phytotoxins in vitro. They can produce already known and novel phytotoxins. ….WHAT’S NEXT? Are the phytotoxins produced in planta ? Are they involved in pathogenicity and virulence of these pathogens? What is their role in the symptomatology observed in vineyards? What gene families are involved in phytotoxin production?

Botryosphaeriaceae spp.: D. seriata (H142a) S. viticola (L19) OBJECTIVE 2: Production and translocation of PMs in artificially-inoculated and naturally-infected plants Botryosphaeriaceae spp.: D. seriata (H142a) S. viticola (L19) Do. vidmadera (L5) Varieties: Chardonnay Cabernet Sauvignon inoculate with mycelia plugs 12 plants per variety-treatment sub-samples will be collected at 6, 9 and 12 months. Naturally infected plants: Tumbaramba and Harden

EXPERIMENTS IN PROGRESS: Isolation Phytotoxins extraction and LC-QqQ analysis DNA extraction and qPCR

OBJECTIVE 3: Investigate the Genetic mechanisms involved in the production of phytotoxins by Botryosphaeriaceae spp. Amplification of (R)-mellein gene, SN477 (Chooi et al. 2015). Designed 7 different primers: NP 1 – NP 7.   DNA extraction  (R)-mellein gene detection  Only NP 1 was amplified for Neofusicoccum parvum, no amplification for Diplodia seriata.  ….WHAT’S NEXT? Purify PCR product and sequence. Design novel primers for D. seriata

ACKNOWLEDGEMENTS